CCK8 full name Cell Counting Kit-8 reagent for simple and accurate cell proliferation and toxicity analysis. The basic principle is: the reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 - benzene disulfonate-2H-tetrazole monosodium salt], which is in the cell under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS) The dehydrogenase is reduced to a highly water-soluble yellow formazan dye. The amount of formazan produced is proportional to the number of living cells. This feature can therefore be used to directly perform cell proliferation and toxicity analysis. The main purposes of this reagent are: drug screening, cell proliferation assay, cytotoxicity assay, tumor susceptibility test

WST-8 is an upgraded product of MTT. Its working principle is: in the presence of electron coupling reagent, it can be reduced by dehydrogenase in mitochondria to produce highly water-soluble orange-yellow formazan product (formazan). The depth of color is directly proportional to the proliferation of cells and inversely proportional to cytotoxicity. The OD value was measured at a wavelength of 450 mM using a microplate reader, indirectly reflecting the number of viable cells. Shanghai Chuangsaidi: for WST-8 water-soluble tetrazolium-8, 193149-74-5, commodity number: CLS-42493-100mg, price 930 yuan

The CCK method is widely used, such as drug screening, cell proliferation assay, cytotoxicity assay, tumor susceptibility test, and biological factor activity assay.

CCK method steps

Experiment 1: Cell activity assay

1. Inoculate a cell suspension (100 μL/well) in a 96-well plate, and pre-culture the plate in an incubator (37 ° C, 5% CO 2 ).

2. Add 10 μL of CCK solution to each well (be careful not to create bubbles in the wells that will affect the OD reading).

3. Incubate the plates in the incubator for 1-4 hours.

4. The absorbance at 450 nm was measured with a microplate reader.

5. If the OD value is not measured temporarily, 10 μL of 0.1 M HCL solution or 1% w/v SDS solution may be added to each well, and the culture plate is covered and stored at room temperature in the dark. The absorbance does not change within 24 hours.

Experiment 2: Cell proliferation - toxicity test

1. Dispense 100 μL of cell suspension in a 96-well plate. The plates were pre-incubated in an incubator for 24 hours (37 ° C, 5% CO 2 ).

2. Add 10 μL of different concentrations of the test substance to the plate.

3. Incubate the plates in the incubator for an appropriate period of time (eg, 6, 12, 24 or 48 hours).

4. Add 10 μL of CCK solution to each well (be careful not to create bubbles in the wells, they will affect the OD reading).

5. Incubate the plates in the incubator for 1-4 hours.

6. The absorbance at 450 nm was measured with a microplate reader.

7. If the OD value is not measured temporarily, 10 μL of 0.1 M HCL solution or 1% w/v SDS solution may be added to each well, and the culture plate is covered and stored at room temperature in the dark. The absorbance does not change within 24 hours.

Note: If the substance to be tested is oxidizing or reducing, replace the medium with fresh medium (removal of the medium and wash the cells twice with medium, then add new medium) to remove the effects of the drug. Of course, in the case where the influence of the drug is relatively small, the medium can be directly subtracted from the blank absorption after the drug is added to the medium. Shanghai Chuangsai provides: 65162-13-2, 1-methoxy-5-methylphenazine methyl sulfate salt [for biochemical research], commodity number: C12-M1442-200MG, price 1974 yuan

The quality of CANSPEC provides excellent performance, and its excellent quality meets the various needs of different experiments. Spot, biochemical reagents, APIs, pharmaceutical intermediates, scientific instruments, laboratory consumables, Shanghai Chuangsai, mass products special promotions, welcome Call us!

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